This invention relates to the incorporation of nucleic acid into cellular systems, to vectors effecting such incorporation, and to microorganisms comprising such vectors.
More specifically, this invention relates to the novel bacteriophage TG1, per se; its genome, or genetic component (DNA), entire and as fragments; and its derivatives (deletion and hybrid variants) thereof which are useful as cloning vectors into organisms, such as bacteria, including in particular phage TG1 host strains, for example, Streptomyces cattleya NRRL 8057, Streptomyces avermitilis MA4990, S. viridochromogenes NRRL 3414, S. garyphalus NRRL 2448, or the like. The resultant modified cells are novel and have utility either as a means of producing the foreign nucleic acid and/or its products through replication of the cells and/or induction and replication of the vector as DNA or as phage, or through the imparting of valuable properties to the cells by virtue of the presence of the foreign nucleic acid therein.
The use of bacteriophages for the cloning of DNA is a well-established procedure; however, the application of this technique to phages of the Actinomycetes is recent. In this regard the following articles and patent disclosures are fully incorporated herein by reference:
(1) "Development of a DNA Cloning System in Streptomyces Using Phage Vectors", J. E. Suarez and K. F. Chater; 6 Cienc. Biol. pp. 99-110 (1981) PA0 (2) "Restriction Mapping of the DNA of the Streptomyces Temperate Phage .0.C31 and Its Derivatives", K. F. Chater, C. J. Bruton and J. E. Suarez; 14 Gene, pp. 183-194 (1981) PA0 (3) "DNA Cloning in Streptomyces: Resistance Genes from Antibiotic-Producing Species", C. J. Thompson, J. M. Ward, and D. A. Hopwood; 286 Nature, pp. 525-527 (1980) PA0 (4) "Dispensable Sequences and Packaging Constraints of DNA from the Streptomyces Temperate Phage OC31", K. F. Chater, C. J. Bruton, W. Springer and J. E. Suarez; 15 Gene, pp. 249-256 (1981) PA0 (5) "A DNA Cloning System for Interspecies Gene Transfer in Antibiotic-Producing Streptomyces", M. Bibb, J. L. Schottel, and S. N. Cohen; 284 Nature, pp. 526-531 (1980) PA0 (6) "DNA Cloning in Streptomyces: A Bifunctional Replicon Comprising pBR322 Inserted into A Streptomyces Phage", J. E. Suarez and K. F. Chater; 286 Nature, pp. 527-529 (1980) PA0 (7) "Cloning and Expression in Streptomyces lividans of Antibiotic Resistance Genes Derived from Escherichia coli.", J. L. Schottel, M. Bibb, and S. N. Cohen; 146 Journal of Bacteriology pp. 360-368 (1981) PA0 (8) "Actinophage DNA", K. F. Chater; 21 Developments in Industrial Microbiology, Chapter 6, pp. 65-74 (1980) PA0 (9) U.K. patent application G.B. No. 2023612A; No. 7919158; published Jan. 3, 1980 PA0 (10) U.K. patent application G.B. No. 2031434A; No. 7928156; published Apr. 23, 1980 PA0 (11) U.K. patent application G.B. No. 2018778A; No. 7913033; published Oct. 24, 1979 PA0 (12) European patent application No. 0036259A2; No. 81300858.8; published Sept. 23, 1981 PA0 (13) U.K. patent application G.B. No. 2046272A; No. 8011000; published Nov. 12, 1980 PA0 (14) U.K. patent application G.B. No. 2069503A; No. 8104473; published Aug. 26, 1981 PA0 (15) European patent application No. 0020147A1; No. 8039178.4; published Dec. 16, 1980 PA0 (16) European patent application No. 0020251A2; No. 80400722.7; published Dec. 10, 1980.
Based upon extensive characterizing studies, the bacteriophage TG1 of the present invention, isolated from soil, was identified as a hitherto undescribed actinophage. The phage is a temperate, i.e., lysogenic phage of Streptomyces cattleya MA4297 (NRRL 8057). The phage has Type B morphology, according to Bradley (David E. Bradley (1967) "Ultrastructure of Bacteriophages and Bacteriocins", Bacteriol. Rev. 31, 230-314). The phage head is icosahedral, having a mean head diameter of about 57 nm; the phage tail, including base plate, is about 128 nm in length; as shown by electron microscopy, FIG. 1. The phage nucleic acid is a linear double-stranded DNA molecule 41.47 kilobasepairs in length having cohesive ends. FIG. 2 is a map indicating the locations of the restriction endonuclease cleavage sites of the phage DNA. The locations are given as the distance of the site in kilobasepairs from the end of the phage DNA nearest the single Pst I site. The map shows all sites for the enzymes Hind III, Kpn I, Pst I, Sma I, Sph I, and Xba I which could be determined by agarose gel electrophoresis and ethidium bromide staining of fragments of 0.2 kilobasepairs and larger. The following enzymes fail to cleave the DNA: BamH I, Nae I, Nar I, Pvu II, Sst I, Sst II, Stu I, and Xho I. Certain phage deletion variants and hybrid phage derivatives are similarly described below. Phage TG1 is described in greater detail below; wherein unambiguous characterizing information relating to isolation, maintenance, propagation of the phage and its nucleic acid, as defined, inter alia, by size and restriction mapping is given. Further, as described below, microorganisms containing TG1 prophage, representative TG1 prophage derivatives and TG1 plasmid derivatives are deposited in a culture collection authorized under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (see Table I).
As disclosed herein, phage TG1, its genome, derivatives, and chimeric DNA molecules comprising TG1 genome, or portions thereof, have utility, inter alia, as cloning vehicles in recombinant DNA procedures.
Phage TG1 has certain properties which make it particularly useful as a DNA cloning vector. Its DNA and derivatives thereof, are readily isolated and amplified by transfection of protoplasts, followed by the lytic growth of the resulting phages (see Example 1, Section J). Deletion mutants retaining these functions, as well as the ability to lysogenize the host, and allowing sufficient space for the insertion of significant quantities (several kilobasepairs) of foreign DNA are readily obtained. In addition, the phage DNA possesses a single cleavage site for the restriction endonuclease Pst I. This site is in a dispensable region of the phage genome. Insertion of foreign DNA at this site does not disrupt the lytic and lysogenic functions of the phage (see Example 1, Sections I and M). Such a unique site suitable for the insertion of DNA is an important feature in a cloning vector, since the probability of obtaining hybrid molecules of the required configuration is greatly increased when only two new DNA junctions need to be formed.
A wide choice of restriction enzymes which can be used for cloning into a vector is of great advantage in recombinant DNA experiments. A large number of restriction endonucleases fail to cleave the phage DNA (see above, and Example 1, Section H.) The unique Pst I site can be converted to a unique site for any one of these enzymes by the use of adaptor molecules (see R. J. Rothstein, L. F. Lau, C. P. Bahl, S. A. Narang and R. Wu in Vol. 68 (1979), Methods in Enzymology, S. P. Colowick and N. O. Kaplan (Eds.), pp. 98-109). Another technique for the conversion of the Pst I cloning site to a different unique site is the insertion of a short fragment containing the desired site. Example 2, Section A, includes a method for the insertion into the Pst I site of a short fragment with a single BamH I site flanked by Pst I sites.
The ability of the TG1 vector and its derivatives to insert into the host DNA is another important property. It allows the construction and stable integration into the host of hybrid vectors carrying genes and novel combinations of genes, which can lead to production of compounds new to the host (e.g., hepatitis B surface antigen, see Example 2), or increased production of normal cellular products by, for example, increased gene dose. Integration is also useful for the identification of cloned genes by genetic complementation of host mutants. Once the lysogen containing the gene is identified, the vector allows the recovery of the gene in large quantity simply by the propagation of phage obtained from the lysogen, followed by the isolation of the phage DNA. Thus, the vector is useful for the production of large quantities of the DNA of the plasmid pACYC177 (see Example 1, Section M).
The host range of the phage is of particular interest. Streptomyces cattleya MA4297 (NRRL 8057) produces the compound thienamycin (see U.S. Pat. No. 3,950,357). Thienamycin has a potent anti-microbial activity against both gram-positive and gram-negative bacteria. It is also effective against bacteria resistant to beta-lactam antibiotics, such as penicillins and cephalosporins, due to the presence of beta-lactamases. Another phage host Streptomyces avermitilis produces the complex of chemically related agents called avermectin, which exhibits extraordinarily potent anthelmintic activity (see R. W. Burg et al; 15 Antimicrobial Agents and Chemotherapy, pp. 361-367 (1979)). The phage can be used for the identification, isolation, and study of the genes and gene products involved in the synthesis and regulation of the synthesis of these important compounds by genetic complementation of mutations in these genes (e.g., for thienamycin, see Example 3).
Certain hybrid derivatives of phage TG1 are useful in other ways. These hybrids, whose construction and properties are described in Example 1, Section M, allow TG1 vector derivatives to replicate as plasmids in Escherichia coli. These hybrid phage-plasmid vectors can be transferred between species by transfection or transformation. This property is extremely useful, since it allows the use of genetic techniques which have been developed for Escherichia coli. For example, genes from S. cattleya may be found capable of complementing mutations in E. coli. Mutations can be introduced into the S. cattleya gene in E. coli, for instance, by the use of transposon mutagenesis; or mutations capable of being suppressed by nonsense suppressors (translational stop codons) might be isolated. The latter mutated genes can be transferred back into S. cattleya and used to isolate mutations capable of suppressing translational stop codons in S. cattleya. Such suppressor mutations are very useful in genetic studies of such an organism. The ability of the vector to replciate in E. coli is also useful in other ways. This property was used for the selection of deletions of vector DNA which were outside the region of the inserted plasmid DNA (see Example 1, Section N). Such deletions are important for the eventual cloning of significant quantities of DNA in the vector.
TG1 and its derivatives can be used in other ways to clone DNA sequences excised using a variety of restriction enzymes appropriate for insertion into the DNA of the particular phage vector. A useful compendium of recombinant DNA techniques is Methods in Enzymology, Vol. 68 (1979), Academic Press, N.Y. Modifications in cloning procedure can include, besides the use of other phage variants and other restriction enzymes: treatment of the restricted vector DNA with phosphatase to remove terminal phosphate groups and thus prevent self ligation of the vector DNA; the use of blunt end ligation following removal of the single stranded ends produced by cleavage with a restriction enzyme with Sl nuclease or synthesis of the complement of such single stranded ends with DNA polymerase; and/or DNA tailing reactions providing cohesive ends between vector and the DNA to be inserted (see Nelson and Brutlag, Methods in Enzymology, Vol. 68 (1979) pp. 41-50).
Transfection frequencies may be improved by altering the described conditions or constituents, or by use of liposome encapsulation of ligated DNA followed by fusion of such liposomes to protoplasts of the recipient bacteria. Such a procedure was recently described by J. F. Makins and G. Holt (Nature 293 (1981) pp. 671-673) for liposome transformation of chromosomal DNA. The procedure used may be that described or variations of it.
Screening of phage clones or lysogens for hybrid phage containing the desired inserted DNA may be carried out in several ways depending upon the phenotypic or genotypic nature of the insert. For example, genes capable of complementing mutants blocked in the synthesis of primary metabolites such as inter alia, amino acids, vitamins, purines or pyrimidines can be detected in the following manner. Specific mutant strains are protoplasted and transfected with the ligated DNA and allowed to regenerate in the absence of any selection. The resulting colonies include non-transfected cells, lysogens formed from transfection by non-hybrid phage, as well as those arising from transfection by hybrid phage. Lysogens containing the desired insert are isolated by plating the regenerated culture on minimal media deficient in a particular nutrient. Colonies arise only from those regenerated lysogens that contain the appropriate DNA insert. A second method that also can easily allow for the detection of the desired insert is to transfect wild type protoplasts with ligated DNA, mix these with wild type germlings and plate the mixture on appropriate plates to allow for plaque formation. The developed plates contain plaques due to religation of the parental phage and those due to phage carrying inserts (essentially the same as a method used for isolating the insertion of plasmid pACYC177 into a TG1 derivative, (See Example 1, Section M). The phage are harvested and a sterile phage stock obtained as described in Example 1, Section B. These lysates are used for a transduction of appropriate mutant strains. Only the phage containing the desired insert are capable of transducing the particular mutation and allow for the formation of a colony.
In the case of mutants blocked in the production of a secondary metabolite, such as an antibiotic, which does not normally have an easily detectable growth phenotype, the phage containing the desired insert are detected by plating lysogens obtained by either of the above described methods on appropriate plates. After allowing a period of time for colonies to develop, the plates are flooded with a culture of an organism sensitive to the antibiotic and further incubated. Lysogens containing an insert with a functioning antibiotic synthetic gene produce the antibiotic and cause the formation of a zone of growth inhibition surrounding the colony (see Example 2).
Phage containing inserts of DNA that do not confer any detectable phenotype, such as ribosomal RNA or transfer RNA, can be detected by either plaque or colony hybridization using complementary DNA as probes.
The efficiency of isolating desired clones can be significantly increased if a method for the direct selection of lysogens is developed. Such a selection can be accomplished using as a vector phage DNA that contains an insert of DNA coding for resistance to an antibiotic (see Examples 2 and 3) or a biosynthetic step. Either selection would eliminate from the analysis, colonies arising from uninfected protoplasts.
Phage clones containing genes for antibiotic synthesis can be used for several other experiments. The phage can be used to transduce wild type, or other antibiotic producing cultures to examine the effect of increased dosage of the particular cloned gene on production levels. The techniques of localized mutagenesis may be employed to isolate mutations in the specific cloned gene thus eliminating the introduction of distant but possibly deleterious lesions into a strain. Such localized mutagenesis will likely result in the isolation of mutations increasing or decreasing the production of antibiotic, and will aid in the improvement of producing cultures and in the genetic analysis of antibiotic synthesis and regulation. Recombinant DNA techniques may also allow for the introduction of high level promoter sequences at an appropriate site to greatly increase the expression of the particular antibiotic synthetic gene. The increased amount of gene product may signficantly increase the productivity of the strain. The cloned gene product may be identified by infection of UV irradiated host cells, followed by infection with the particular hybrid phage. Growth in the presence of radio-labelled amino acids would result in the synthesis of radio-labelled proteins coded for by the phage. Proteins coded for by the host cell would not be synthesized due to the damage caused by the UV treatment. The proteins can be separated and analyzed by standard techniques.
Foreign DNA, that is, DNA from sources other than the host organism, can also be inserted into the phage vector. The insertion of pACYC177 into the phage is one example of this. In such cases the presence of a host restriction system may greatly lower the frequency of transfection and thus make detection of desired clones difficult. Bacterial mutants defective in host restriction may be isolated to eliminate this problem. Such a strain and its derivatives can then serve as the host for cloning experiments. Examples of foreign DNA that may prove to be useful include, antibiotic resistance genes, genes coding for informational suppressors (transfer RNA), or mutagenic elements such as transposons or mutator genes.
Cloned antibiotic genes may be physically isolated using standard recombinant DNA techniques and transferred to other cloning vectors. These may include plasmids that replicate in the host organism or those that replicate in other strains. Cloned antibiotic genes may be transferred via TG1 or its derivatives to other Streptomyces strains that normally do not produce the antibiotic. This transfer may result in the synthesis of new antibiotics, or altered expression of the cloned gene.
The vectors can be used in several other ways to achieve DNA cloning. Normally a plasmid of an unrelated bacterial strain, for instance a plasmid of E. coli, if introduced into a phage host strain, will not replicate or be maintained because of its foreign origin. If, however, the plasmid has been altered by the insertion of a DNA sequence homologous to one present in the host chromosome, a recombination event can result in the integration of the plasmid into the chromosome at the site of homology. If the host strain is a phage lysogen, the homologous sequence on the plasmid can be provided by a phage fragment. In this case the plasmid will insert into the prophage which is integrated in the chromosome. Such an inserted plasmid will be maintained as a part of the host chromosome as long as the prophage remains integrated. Alternatively, a specific phage fragment carrying functions necessary for the normal integration of the phage, that is, the attachment site and phage genes coding for gene products necessary for phage integration, can be inserted into the plasmid. Such a vector should be capable of integrating into the host chromosome at the same site as, and in a manner analogous to, the integration of the wild type phage. Recombinant bacteria carrying the inserted vectors can be easily identified if a selectable genotype is also carried by the vector. Vectors of this type have several important advantages: (1) the amount of DNA which can be cloned into such a molecule is not limited by phage packaging constraints; (2) the plasmid can replicate in its orginial host, for instance E. coli, where various genetic and physical manipulations may be easier to achieve than in the Streptomyces host; (3) an element, such as the lambda cos site can be introduced, allowing cloning of large DNA fragments by the technique of cosmid DNA cloning (see Methods in Enzymology, Vol. 68) in the plasmid host, e.g., E. coli, followed by isolation of the resulting hybrid DNA and transformation or transfection of protoplasts of the Streptomyces strain.
Other phage sequences may also be used for cloning purposes. One such sequence is the cos site of phage TG1. This site is recognized by a phage product during packaging of DNA into phage capsids. A minimal length of DNA is required between successive cos sites for efficient packaging. If the cos site is inserted into a plasmid and other DNA is also inserted then an in vitro packaging system can be used to process the DNA and form phage particles. Only those inserts that create the correct length of DNA between cos sites can be packaged and thus large inserts are selected.
Phage promoter sequences can also be utilized to place chromosomal genes under different controls. Some of the phage promoters are regulated by phage regulator mechanisms. Thus expression of properly inserted cloned genes will also be controlled by such mechanisms. For example, a temperature sensitive repressor may, at low temperature, greatly lower the expression of a particular promoter by binding to an operator site, at elevated temperature the repressor may be inactive and transcription at high levels may occur. If a desired cloned gene were inserted at an appropriate position, its expression and the level of the gene product could be controlled during a fermentation by adjusting the temperature.
TABLE I ______________________________________ Microorganism Deposit Strain Resident Accession Number.sup.a Vector Number.sup.b ______________________________________ MA5766 TG2 ATCC 39077 MA5767 TG1 ATCC 39078 MA5769 TG4 NRRL 15034 MA5770 TG5 NRRL 15035 MB4540 pTG6 ATCC 39079 MB4541 pTG12 ATCC 39080 MB4542 pTG16 ATCC 39081 MB4543 pTG7 ATCC 39082 MB4544 pTG15 NRRL B-15027 MB4545 pTG8 NRRL B-15028 MB4546 pTG9 NRRL B-15029 MB4547 pTG10 NRRL B-15030 MB4548 pTG11 NRRL B-15031 MB4549 pTG13 NRRL B-15032 MB4550 pTG14 NRRL B-15033 ______________________________________ .sup.a Strain designations are those of the culture collection of MERCK and CO., Inc., Rahway, New Jersey. MA5766-MA5770 are derivatives of Streptomyces cattleya MA4297 (NRRL 8057) lysogenic for the indicated phag vectors. MB4540-MB4550 are derivatives of Escherichia coli K12 RR1 containing the indicated hybrid phageplasmid vectors. E. coli K12 RR1 is described by F. Bolivar, R. L. Rodriquez, M. C. Betlach, and H. W. Boyer; 2 Gene, pp. 75-93 (1977). .sup.b Cultures of each strain have been placed on permanent deposit with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., and were complete as of April 2, 1982; and at the Agricultural Research Culture Collection (NRRL), Fermentation Laboratory, Northern Regional Research Center, Science and Education Administration, U.S. Department of Agriculture, 1815 North University Street, Peoria, Ill., as of April 2, 1982; and were assigned the indicated accession numbers.